Oral insulin and a method of making the same

ABSTRACT

The present invention provides for a composition of matter useful as an oral dosage form of insulin based upon a two phase liquid aqueous system in which insulin component or components are incorporated. The invention also provides for a method of preparing the oral dosage form of insulin, as well as a further process whereby sustained release dosage forms of oral insulin are produced. The oral form of insulin eliminates or reduces the need to use injection as a mode of administering insulin.

This application is a continuation-in-part of U.S. Ser. No. 608,483filed May 9, 1984.

This invention concerns an improved form of insulin which is orallyadministered, and methods of preparation.

Review of the scientific literature indicates that despite intensiveresearch, the problems obstructing the development of an oral dosageform of insulin remain unsolved. The published research includes noreport of a delivery system that will make it possible to administerinsulin orally. (Reference: Chemical Abstracts 1970-1984)

The investigations of Dapergolas and Gregoriadis suggest that amoderately effective hypoglycemic effect may be achieved through theintragastric use of liposome entrapped insulin. Examination of the dataof these experiments indicate that the rate of insulin absorption waserratic and further, that it was not possible to determine the quantityof insulin that was absorbed. (Reference: Dapergolas, G. andGregoriadis, G. Hyperglycemic Effect of Liposome Entrapped InsulinAdministered Intragastrically Into Rats; Lancet, Oct. 16, 1976, pp.824-827)

The use of liposomes to prepare an oral form of insulin is based upon aconcept and a method manufacture that differs fundamentally from theconcept and method of the herein disclosed invention. A two phasecoacervate system comprises the basis of this invention; it bears norelationship to liposomes. Moreover, given the known difficulties in themanufacture and use of liposomes, such compositions do not appear to bethe basis on which a useful oral insulin may be developed. (Reference:Ostro, Marc, Lipsomes, Marcel Dekker, New York, 1983)

The advantages of an oral dosage form of insulin are readily apparent.An oral form of insulin will eliminate or reduce the need to useinjection as the mode of administering insulin. Further, it appears thatan oral form of insulin could be used to advantage in the treatment ofadult onset diabetes mellitus. The sulfonylureas are now prescribed forthe treatment of this form of diabetes despite the adverse effectsassociated with the use of this class of drugs. The sulfonylureas arenot considered to be an acceptable substitute for insulin nor are theyconsidered to be oral forms of insulin.

It is important to note that no known oral dosage form of insulin hasbeen developed since the discovery of insulin more than sixty years ago.

The present invention discloses an oral dosage form of insulin and amethod by which said insulin can be prepared. However, it should benoted that the herein disclosed invention constitutes a safe, effectiveoral dosage form not only for insulin but for non-polar drugs and othermedical compositions known to be subject to degradation in the digestivesystem.

Examples of the applicants' discovery of coacervate systems by means ofwhich drugs, enzymes, nutrients and hemoglobin may be introduced intoand transported in the circulatory system are disclosed in U.S. Pat.Nos. 4,343,797 and 4,439,424. In the present disclosure, the concepts ofthe inventions referred to above have been modified and utilized in amanner that yields an oral as opposed to an intravenous preparation.Moreover, the specific use of the coacervate system of this inventiondistinguishes, the claimed composition and its method of preparationfrom the known microencapsulated biologicals and drug products and themethods used to produce them. At this point, the state of the art doesnot include an oral composition which will protect drugs, biologicalsand other medical preparations vulnerable to degradation by enzymes, pH,acid-base balance and other conditions and processes of thegastrointestinal tract.

It is an object of this invention to provide a delivery system thatenables the safe, effective oral administration of the known forms ofinsulin. When insulin is incorporated in the herein disclosed deliverysystem it retains all of its known pharmacologic and physiologicalproperties. Furthermore, this invention provides a convenient method ofpreparation of the claimed oral dosage forms.

The invention provides a composition of matter useful as an oral dosageform of insulin; said preparation comprising a non-toxic two phaseliquid system, both phases being aqueous;

(a) one of said phases being a relatively nonpolar coacervate phase;

(b) The other of said phases being a relatively polar liquid aqueousphase;

(c) said relatively non-polar coacervate phase being insoluble in and inequilibrium with said relatively polar liquid aqueous phase;

(d) said two phase system, when prepared to contain an insulin componentor components in the coacervate phase of the coacervate system, yields adelivery system for the oral administration of insulin.

Moreover, the invention provides for a method of preparing a compositionuseful as an oral dosage form of insulin, said method based upon the twophase liquid system. A preferred method is characterized by the steps of(a) combining albumin and lecithin in equal proportions in sterilewater; (b) thoroughly mixing the components; (c) storing said mixtureundisturbed until the composition of step (a) separates into two layers,one above the other, the lower phase being a substantially non-polarcoacervate phase, the upper phase being an equilibrium water phase; (d)continuing the separation process until no increase in the volume of thecoacervate system can be observed; (e) centrifuging the compositionuntil inspection reveals a clear demarcation of the two phases and (f)separating the two phases.

Given that no oral dosage form of insulin now exists, the hereindisclosed invention represents a significant scientific and medicaladvance. By using any one of a number of appropriate non-toxiccoacervate systems as a beginning step in the manufacture of thisinvention, it becomes possible, through a subsequent sequence ofrelatively simple processing steps to produce a delivery system for theoral administration of insulin. The said system can be produced as aliquid or as an encapsulated composition.

In this disclosure, the term particle refers to encapsulated or emulsiondroplet entities. In the claimed invention, the size of the particleswhich will contain the hemoglobin component may range from nanometersize to micron size; from 10⁻⁸ microns to 10 microns. Particles in thisrange of sizes are known to pass through the wall of the small intestineand, it follows, into the circulatory system. There is reason to believethat in the finished product of this invention, the coacervated microand sub-micro encapsulated particles will tend to adhere to the membraneof the gastrointestinal tract, thereby facilitating passage through thewall of the small intestine.

Another singularly important feature of this invention is the protectionafforded the insulin component of this composition from enzymaticdegradation in the digestive tract. This is accomplished byincorporating the insulin in the coacervate phase of the coacervatesystem. In the process of this incorporation, a film of coacervate phasewater surrounds and coats each insulin molecule. This feature is offundamental importance since coacervate phase water differs in physicalchemical structure from the bulk water present in the stomach. Thedigestive enzymes which degrade insulin readily diffuse in theaforementioned bulk water, however, since these enzymes diffuse onlyminimally, if at all, in the water of the coacervate phase. The rate ofinteraction between the insulin component and the digestive enzyme isextremely slow. Since the rate of interaction is so slow,physiologically useful quantities of insulin escape destruction, passthrough the wall of the small intestine and into the circulation.Moreover, in this invention, the insulin component is protected from thedegradation effects of pH, acid-base balance and other conditions andprocesses of the gastrointestinal tract.

In the manufacture of the claimed composition, any of the known forms ofinsulin, i.e., regular insulin, globin zinc insulin, insulin zincsuspension, prompt insulin zinc suspension, extended insulin zincsuspension, or combinations thereof may be used. The insulins used inthis invention may be of U.S.P. standard, if desired. Non U.S.P.insulins may be used provided they are acceptable in other fundamentalrespects.

If preferred, the insulin component may be comprised of recrystallizedinsulin obtained from animal, recombinant genetic, synthetic or othersources. Since the claimed compositions are intended only for oral use,the insulin used in this invention need not be subjected to the rigorousmanufacturing steps required for U.S.P. injectable insulin. However, ifdesired, insulin prepared for injection may be used in this invention.

The claimed composition may be prepared to any of severalspecifications. Thus, it may consist of insulin dispersed in thedisclosed coacervate system and dispensed as a liquid preparation. Themethod of this invention also provides for additional steps in which thepreviously described preparation is further processed to yield a productwhich is comprised of particles of encapsulated insulin. The particlesare within the size range referred to previously. The final product canbe placed in conventional gelatin capsules and dispensed as such, oralternatively, the particles containing insulin can be dispensed in anysuitable liquid vehicle, i.e., a vehicle based on coacervate phase waterand dispensed for use in that form. The invention provides for sustainedrelease dosage forms and for emulsion and suspension preparations.

Any appropriate non-toxic coacervate system can be used in themanufacture of the product of this invention. Further, any endogenousbiological surface active agent or derivatives thereof, e.g., albumin,lecithin, gelatin, etc., can be used to make a coacervate systemappropriate for the product and process of this invention.Alternatively, suitable non-toxic exogenous components can also be usedto prepare the coacervate system, for example, acacia when combined withgelatin. Many other surface active agents and/or combinations thereofthat can form multiple coacervate phase systems can also be used.However, it is fundamental to the claims of this invention thatregardless of the constituent ingredients used, the first manufacturingstep involves this preparation of an appropriate coacervate system. Uponcompletion of this step, the coacervate system will consist of twophases: (1) an internal suspension, relatively non-polar phase, commonlyreferred to as the coacervate phase, and (2) an associated, relativelypolar, external suspension or equilibrium phase. These phases areinsoluble in and in equilibrium with each other. The coacervate phase ofthe disclosed coacervate system can comprise from 0.5 to 99.5% by volumeof the system; correspondingly, the associated equilibrium phase cancomprise from 0.5 to 99.5% by volume of the herein described coacervatesystem.

In order to explain the claimed composition and method of thisinvention, the following is a description of the preferred method ofmanufacture of said composition and the composition itself. Specificexamples which follow will explain and illustrate the variety offormulations that are possible within the parameters of this disclosure.

As noted previously, preparation of an appropriate coacervate systemcomprises the first step in the manufacture of the claimed compositionof matter. The said coacervate system may be formulated using anyacceptable non-toxic biological, non-biological surface active agent,derivatives or mixtures thereof. Sources of the surface active agentcomponent may be either of natural or synthetic origin. Any of thefollowing are examples of acceptable surface active agents: albumin,suitable phospholipids, gelatin, modified fluid gelatin, acacia gel andother surface active compositions known to those skilled in the art. Inthis invention, appropriate combinations of these agents are preferred.

In the method of this invention, the combination of albumin and aphospholipid, preferably lecithin is preferred. Other phospholipids suchas cephalin, isolecithin, sphingomyelin, phosphatidyl serine,phosphatidic acid, phosphatidyl inositol and phosphatidyl choline may beused in place of the preferred lecithin.

In the preferred method of this invention, equal weight to volumeproportions of albumin and lecithin are added to a quantity of sterilewater that will yield 100 mls of aqueous solution. Any quantity ofalbumin and lecithin can be used, provided that the proportions givenabove are observed and the quantity of water is adjusted accordingly.Three per cent weight to volume of lecithin and three per cent weight tovolume of albumin is preferred. Unequal proportions of thesecompositions may be used to produce a coacervate system; however, whilesuch systems may have useful characteristics, unequal proportions arenot believed to be preferable i.e.; drug transport, etc., for thisinvention.

In the method of this invention, other combinations of surface activeagents may be used. These combinations would include the following:acacia gel and gelatin or modified gelatin, two gelatins of the same ordiffering isoelectric points, two modified fluid gelatins of the same ordiffering isoelectric points and other combinations of surface activeagents known to those skilled in the art.

Following the step in which albumin and lecithin are added to sterilewater, the solution is thoroughly mixed and stored, undisturbed insuitable containers. The storage step takes place at a temperature whichcan range from the freezing point to 4° C. to room temperature. In thedisclosed preferred method storage takes place at any temperature withinthe range of 4° to 15° C.

During the period of storage, the solution described above will separateinto two phases. The upper phase (layer) is referred to as theequilibrium water phase. The lower phase (layer) is referred to as thecoacervate phase. The period of storage is continued until the maximumyield of the coacervate phase of the coacervate system has beenachieved. Maximum yield is the point in the manufacturing process atwhich no significant increase in the volume of the coacervate phase canbe observed. This determination may be made by visual inspection orother suitable means. As is known to those skilled in the art, longerperiods of storage will produce greater yields of the coacervate phase.

When it is determined that the maximum yield of the coacervate phase hasbeen achieved, the coacervate system is centrifuged until observationindicates that a clear division exists at the interface of the twophases of the coacervate system. At this point, the two phases areseparated from each other by means of a separatory funnel. The desiredamount and type of insulin is then added to the coacervate phase andmixed thoroughly.

Any of the known forms and dosages of insulin, i.e., regular insulin,globin zinc insulin, insulin zinc suspension, prompt insulin zincsuspension, extended insulin zinc suspension, protamine zinc insulin orcombination thereof may be used. The insulin(s) used may be of U.S.P.standard. If preferred, the insulin component may be one directlyderived from recrystallization of insulin from animal, recombinantgenetic or other sources. The invention contemplates the manufacture ofa variety of oral insulin preparations, compositions and dosages inorder to meet specific medical requirements. Accordingly, no singleinsulin specification can be described as preferred. For purposes ofthis disclosure, the individual dose can be 100 standard units and/orfractions and/or multiples thereof.

After the step in which the insulin component is dispersed in thecoacervate phase and thoroughly mixed, the preparation may be dispensedas an oral dosage form of insulin. Preferably, the manufacturing processis continued. In that case, the next procedure involves therecombination of the equilibrium water phase and the coacervate phasewhich now contains the insulin component.

Following the recombination of the phases, the preparation is emulsifiedusing a colloid mill, sonifier or other emulsifying technique known tothose skilled in the art. The particles of the emulsion may range insize from 10⁻⁸ microns to 10 microns. It is preferred that the particlesize be within the nanometer and millimicron range.

The product resulting from the emulsifying step comprises a promptrelease form of insulin and may be used as such or stored underrefrigeration until needed. The emulsified composition is, if desired,processed further to produce time release, i.e., sustained release formsof insulin(s). To produce these forms of insulin, the preparation issubjected to a heating step, i.e., the preparation is heated for from 30seconds to 15 minutes at a temperature which may range from 20° to 70°C. Since this step will harden the surface film surrounding eachparticle, the variables of time and temperature may be varied to producea spectrum of particle surface film hardnesses. This method comprisesthe preferred method. Other processes may be used to prepare sustainedrelease forms of the claimed composition. Thus, a cross linking agent,such as a non-toxic entity within the aldehyde group, i.e.,gluteraldehyde may be used in a chemical process which harden the filmsurface of the particles containing the insulin component.

As a result of the heating step, the claimed oral insulin composition iscomprised of particles which are characterized by differing degrees ofsurface film hardness and thus, differing rates of time release. Afterthe heating step is completed, the composition is filtered through afilter pad. The product remaining in the filter bed constitutes theparticles of oral insulin. These are removed from the filter bed by awashing step and then dried by any acceptable drying process. Uponcompletion of the drying step, the preparation of particles containingoral insulin is completed. The composition may now be placed in anappropriate oral dosage form. Thus the desired dose of the compositionmay be resuspended in a suitable non-toxic liquid vehicle, or placed inother conventional oral dosage forms and dispensed or stored, preferablyunder refrigeration until needed. If it is desired, an appropriate doseof the prompt release particles may be mixed with an appropriate dose ofthe previously described time release oral insulin(s) formulation. Thismixture is then placed in any acceptable, non-toxic oral dosage form anddispensed or stored, preferably under refrigeration until needed.

Previously, in this disclosure it was stated that a number of entitiesmay be used to prepare the required coacervate system. An example of analternative acceptable coacervate system for preparing oral insulin isbased on the use of gelatin. In this alternative, the following stepsare used to produce the coacervate system. Five per cent gelatinsolution is dispersed in distilled water and heated to 40° C. 0.9%sodium chloride solution is slowly added to the preparation until thesolution separates into two layers. The lower layer comprises thecolloid rich liquid gelatin or coacervate phase. The upper layerconsists of the equilibrium water phase.

The two phases are separated by means of a separatory funnel. Thedesired quantity and type of insulin or combinations thereof, asdescribed above, are then added and mixed into the coacervate phase, andmay, if desired, be dispensed as oral liquid insulin. If preferred, themanufacturing process proceeds and the two previously separated phases,one of which now contains insulin, are recombined. Through the use ofany acceptable emulsifying technique, an emulsion consisting ofnanometer, millimicron and micron size particles inside of which insulinis contained is prepared. The emulsion is simultaneously rapidly cooledand stirred at a temperature of from 0° to 10° C. for from 15 to 60minutes by means of a refrigerated stirring unit. After this stirringstep, the composition is forced through a filtering system. The productremaining in the filter bed is removed through a washing step. Uponcompletion of this step the particles are dried in an air drying system.When the encapsulated particles of insulin are completely dried, thequantity and type of insulin necessary for a specific dose is placed inany appropriate oral drug delivery system. The conventional gelatincapsule is preferred.

SPECIFIC EXAMPLES

Examples of how the claimed composition(s) of matter may be preparedfollow:

EXAMPLE 1

5% weight to volume proportions of albumin and lecithin are added to anamount of distilled water that will yield 100 mls of aqueous solution.The mixture is then thoroughly mixed by a vortex mixer. Following themixing step, the solution is stored undisturbed until the maximum yieldof the coacervate phase of the coacervate system has been achieved. Thestorage step takes place at 4° C. When it is observed that the maximumyield of the coacervate phase has been achieved, the coacervate systemis centrifuged until observation indicates that a clear division existsat the interface of the two phases of the coacervate system. The twophases are then separated by means of a separatory funnel. Theequilibrium water phase resulting from this separation step is set asidefor subsequent recombination with the coacervate phase. Next, 100standard units of regular insulin U.S.P. is thoroughly mixed into thecoacervate phase. Following this step, the two phases are recombined andsubjected to an emulsifying procedure to produce an emulsion, theparticles of which will range from the nanometer to the millimicronsize. The emulsified product comprises a prompt release form of insulinand may be used as such or stored, preferably under refrigeration, untilneeded.

EXAMPLE 2

The method of Example 1 is followed except that Extended Insulin ZincSuspension U.S.P. is used in place of regular insulin and the particlesremoved from the filtration bed are placed in gelatin capsules in dosesthat are medically appropriate.

EXAMPLE 3

The method of Example 1 is followed except that Prompt Insulin ZincSuspension U.S.P. is used in place of regular insulin.

EXAMPLE 4

The method of Example 1 is used except that Globin Zinc InsulinSuspension U.S.P. is used in place of regular insulin. A mixture of theproduce of the emulsification step and the product subjected to theheating step is prepared and dispensed in an appropriate liquid dosageform.

EXAMPLE 5

The method of Example 1 is used except that Protamine Zinc Insulin isused in place of regular insulin.

EXAMPLE 6

The method of Example 1 is used except that a mixture of regular insulinU.S.P. and Insulin Zinc Suspension U.S.P. are used in place of regularinsulin.

EXAMPLE 7

200 mls of 5% solution of albumin is added to 200 mls of 3% solution oflecithin and mixed thoroughly. The remaining steps of the procedurefollow those of Example 1, except that Protamine Zinc Insulin are usedin place of regular insulin.

EXAMPLE 8

200 mls of a 5% solution of albumin is added to 200 mls of a 7% solutionof lecithin and mixed thoroughly. The remaining steps of the procedurefollow Example 1, except that Globulin Zinc Insulin Suspension U.S.P. isused in place of Regular Zinc Insulin.

EXAMPLE 9

200 mls of a 3% solution of albumin is thoroughly mixed with 200 mls ofa 3% solution of isolecithin. The solution is then stored undisturbed at4° C. for 24 hours. The remaining steps of the procedure follow Example1.

EXAMPLE 10

Disperse 6% gelatin solution in distilled water and heat to 40° C. Next,slowly add that amount of sodium chloride to the solution as will resultin a two phase system. The lower layer comprises the colloid rich liquidgelatin phase, the upper layer constitutes the coacervate phase. The twophases are separated and 100 units of regular insulin U.S.P. is added tothe coacervate phase. Recombine the two phases, mix thoroughly andthrough the use of a colloid mill prepare an emulsion of the recombinedphases wherein the size of the particles of said emulsion are in therange of 10⁻⁸ microns. Following the emulsifying step, the emulsion issimultaneously rapidly cooled and stirred at a temperature of 5° C. for30 minutes. The resulting particles are thoroughly rinsed with distilledwater following which they are completely dried. The quantity of thecomposition placed in gelatin capsules is a function of the medicallyindicated dosage of insulin or combinations thereof. The particles arethen placed in gelatin capsules in the dosages and types of insulindesired.

EXAMPLE 11

The method of Example 9 is followed except that Isophane InsulinSuspension U.S.P. is used in place of regular insulin U.S.P.

EXAMPLE 12

The method of Example 1 is followed except that Extended Insulin ZincSuspension U.S.P. is used in place of Regular Insulin U.S.P.

EXAMPLE 13

The method of Example 1 is followed except that Lente Insulin is used inplace of regular insulin.

EXAMPLE 14

The method of Example 1 is followed except that recrystallized insulindirectly derived from animal sources is used in place of Regular InsulinU.S.P.

EXAMPLE 15

The method of Example 1 is used except that a combination of regular andglobin zinc insulin are used in place of regular insulin.

EXAMPLE 16

200 mls of a 3% solution of albumin is thoroughly mixed with 200 mls ofa 3% solution of isolecithin. The solution is then stored undisturbed at4° C. for 24 hours. The remaining steps of the procedure follow Example1, except that Prompt Insulin Zinc Suspension U.S.P. is used in place ofregular insulin U.S.P.

EXAMPLE 17

The method of Example 1 is used except that a combination of RegularInsulin U.S.P., Prompt Zinc Suspension and Extended Insulin ZincSuspension is used in place of regular insulin.

EXAMPLE 18

The method of Example 1 is used except that after the two phases of thecoacervate system are separated, 10 units of regular insulin aredispersed into the coacervate phase and mixed thoroughly. Thecomposition is then dispensed as oral liquid insulin or placed inrefrigerated storage until it is to be dispensed.

EXAMPLE 19

The method of Example 10 is followed except that after two phases of thesystem are separated 20 units of insulin are dispersed in the coacervatephase and mixed thoroughly. The preparation can either be dispensed asan oral liquid insulin preparation or stored until needed.

EXAMPLE 20

Example 1 is followed except that the final product of said example isheated for 20 seconds at a temperature of 40° C. Following the heatingstep, the composition is forced through a filter bed. The productremaining in the filter bed is removed through a washing procedure andthen dried. Upon completing of the drying step, the composition may beplaced in any acceptable oral dosage vehicle in the indicated dose.

EXAMPLE 21

Example 1 is followed except that the heating step is carried out for 10seconds at 35° C.

EXAMPLE 22

Example 1 is followed except that 0.5% weight to volume ofgluteraldehyde is added to the final product of said example. Thecomposition is mixed thoroughly and then subjected to a filtering step.The particulate matter remaining in the filter bed is removed and placedin refrigerated storage for three to five hours, after which thecomposition is washed with distilled water until no trace of thegluteraldehyde is found in the wash water. This step is followed by aperiod of drying, after which the product of the drying step may beplaced in any suitable dose in any conventional oral dosage vehicle.

EXPERIMENT

Two white laboratory rats, weighing 177 and 197 grams, respectively,were used in this experiment. One rat was administered a preparation oforal insulin according to the present invention containing 5 units ofinsulin; a formulation of the oral insulin according to the presentinvention containing 10 units of bovine insulin was administered to thesecond rat. Blood samples were drawn from each animal before and threehours after administration of the oral insulin preparation. The resultsgiven below are expressed as the percentage of the pre-experiment bloodglucose level.

    ______________________________________                                                Units of                                                              Animal  Bovine Insulin  Result                                                ______________________________________                                        1        5              66.3% of initial blood                                                        glucose level                                         2       10              47.1% of initial blood                                                        glucose level                                         ______________________________________                                    

One white laboratory rat weighing 181 grams received a non-encapsulatedpreparation containing 5 units of free bovine insulin. This animalserved as the control. After three hours, blood glucose level was 98.7%of the pre-experiment blood glucose level.

I claim:
 1. A method of introducing insulin into a circulatory system comprising orally ingesting a composition comprising an aqueous coacervate system including water, a surface active agent and an effective amount of insulin, said coacervate system including an aqueous coacervate-based film encapsulating the insulin; said aqueous coacervate-based film comprising an aqueous colloid-rich phase, or an aqueous equilibrium water phase, or a combination thereof.
 2. A method of adding insulin to the circulatory system of a mammal comprising having the mammal orally ingest a composition comprising an aqueous coacervate phase derived from a two-phase coacervate system, said coacervate phase including water, a surface active agent and an effective amount of insulin, said aqueous coacervate phase including an aqueous coacervate-based film encapsulating the insulin.
 3. A method of treating diabetes comprising having a diabetic orally ingest an effective amount of a composition comprising an aqueous coacervate phase derived from a two-phase coacervate system, said coacervate phase including water, a surface active agent and an effective amount of insulin, said aqueous coacervate phase including an aqueous coacervate-based film encapsulating the insulin. 